Paraformaldehyde-fixed, paraffin embedded (rat skeletal muscle); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (p-MAP4K1(Ser171)) Polyclonal Antibody, Unconjugated (bs5494R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Molt-4 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 20% PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-MAP4K1 Antibody(bs-5494R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).